Golgi associated Rab proteins - small GTPases of the Ras superfamily -- regulate critical, yet poorly understood, aspects of Golgi biogenesis, maintenance, and inheritance through affecting vesicle trafficking. The general (i.e., "textbook") paradigm holds that Golgi Rab proteins regulate vesicle targeting via the recruitment and activation of tethering factors that mediate initial steps of membrane fusion. Our Preliminary Studies support a different and novel mechanism. We screened for molecular genetic relationship(s) between Rab33b and Rab6 - two Golgi Rabs that our works implicates in an intra-Golgi Rab cascade -- and the putative tether proteins, COG and ZW10/RINT-1, central to retrograde Golgi trafficking. We found that both Rab6 and Rab33b acted upstream of, not through, the tether complexes. By electron microscope tomography (ET), we observed that Rab6 knockdown results in an apparent inhibition of Golgi vesicle budding/transport as evidenced by the pronounced accumulation of both coated and uncoated budding profiles/vesicles along Golgi cisternal membranes. This has led us to conclude that Rab6 is required for efficient Golgi vesicle release/scission. In sum, our results suggest that Golgi Rabs such as Rab6 and Rab33b serve important functions in very early events at the Golgi related to vesicle scission, and that they act upstream of tether factor recruitment in this fundamental process. Based on our Preliminary Studies, we thus contend that a critical and functionally important role of Golgi-associated Rabs (such as Rab6 and Rab33b) is to regulate, through context sensitive effector sets, the budding/release of distinct classes of transport vesicles from Golgi cisternae. Functionally, we predict that each vesicle class supports a distinct trafficking pathway(s) and as such are compositionally distinct. Cellularly, we hypothesize that cisternal Rab proteins such as Rab6 and likely Rab33b play an important/central role in Golgi homeostasis. This hypothesis is supported by our finding that compared to control cells, Rab6 depleted cells reproducibly reveal a significant increase in the number of Golgi cisternae per stack (4.2 + 0.32 versus 6.8 + 0.46, respectively). We propose that Rab proteins regulate the distribution of Golgi resident proteins between cisternae. We predict that in Rab knockdown experiments that the distribution of individual Golgi enzymes will be shifted cis- or trans-ward. Mechanistically, we hypothesize that distinct effectors, including already identified candidate protein and others novel, will modulate the budding of individual vesicle classes. We propose the following three Specific Aims to test these hypotheses. Specific Aim 1. We will test the hypothesis that Golgi associated Rab proteins such as Rab6 and Rab33b regulate the formation and/or release of multiple, distinct classes of vesicles from Golgi cisternae. Specific Aim 2. We will test the hypothesis that Golgi-associated Rabs regulate the distribution of Golgi resident proteins between cisternae. Specific Aim 3. We will test the hypothesis that mechanistically distinct protein sets, i.e., effectors, modulate the budding/release of individual vesicle classes. Characterization of structural/functional relationships within the mammalian Golgi apparatus and its regulatory pathways is important to human health. We and other investigators have found that such pathways are useful portals into the cell for the delivery of cell-killing reagents and antigens. Defects in these pathways can lead to human disease. Moreover, important machinery components in these pathways are involved in such processes as virus infection and aging. Modulation of Golgi associated Rabs may prove to be an important therapeutic approach. PUBLIC HEALTH RELEVANCE: The Golgi apparatus is the central organelle within the secretory pathway of human cells. How the structure and function of this organelle is regulated is central to the health of cells and hence to the health humans. We find that Rab proteins, small GTPases of the Ras superfamily, regulate Golgi organization, likely at the level of vesicle formation/release/transport, a surprising outcome. Defects in Golgi associated Rab protein function have been already shown to affect vision (glaucoma), neurodegenerative disease, Chlamydial infection, viral entry and aging. Retrograde trafficking through the Golgi apparatus can be used as a portal for drug and antigen delivery. Rab modulation by small molecules may be a novel approach to controlling infection and other disease problems. Understanding the role of Rab proteins in structure and function relationships within the human Golgi apparatus should indeed further our understanding of human health.